Our above-mentioned copending U.S. patent application, the entire content of which is incorporated by reference as though set forth herein, discloses and claims a new synthesis of stable, water-soluble chemiluminescent 1,2-dioxetanes, particularly ones that are enzymatically cleavable, substituted with stabilizing and solubilizing groups and ring-containing fluorophore moieties. The synthesis employs dialkyl 1-alkoxy-1-arylmethane phosphonate .alpha.-carbanion intermediates in the synthesis of key enol ether intermediates for the desired 1,2-dioxetane end products.
Among the 1,2-dioxetanes that can be obtained by the novel synthetic method of our above-mentioned application are those represented by the formula: ##STR2## In this formula the symbol T represents a spiro-bonded stabilizing group, a gem carbon atom of which is also the 3-carbon atom of the dioxetane ring.
The most preferred stabilizing groups represented by T are fused, substituted or unsubstituted polycycloalkylidene groups, bonded to the 3-carbon atom of the dioxetane ring through a spiro linkage and having two or more fused rings, each ring having from 3 to 12 carbon atoms, inclusive, e.g., an adamant-2-ylidene group. The fused polycycloalkylidene group may additionally contain unsaturated bonds or 1,2-fused aromatic rings, or a substituted or unsubstituted alkyl group having from 1 to 12 carbon atoms, inclusive, such as methyl, ethyl, tertiary butyl, trifluoromethyl or 2-cyanoethyl, or an aryl or substituted aryl group such as carboxyphenyl, or a halogen group such as fluoro or chloro, or a heteroatom group which can be a substituted or unsubstituted alkoxy or aryloxy group having from 1 to 12 carbon atoms, inclusive, such as a methoxy, ethoxy, hydroxyethoxy, methoxyethoxy, carboxymethoxy, or polyethylethyleneoxy group, or a cyano group, a methanesulfonyl group or other electron-withdrawing group.
OR.sup.3 is an ether group, prefereably a lower alkyl ether group such as a methoxy group, in which the symbol R.sup.3 represents a C.sub.1 -C.sub.20 unbranched or branched, substituted or unsubstituted, saturated or unsaturated alkyl group, e.g., methyl, ethyl, allyl or isobutyl; a heteroaralkyl or aralkyl (including ethylenically unsaturated aralkyl) group, e.g., benzyl or vinylbenzyl; a polynuclear (fused ring) or heteropolynuclear aralkyl group which may be further substituted, e.g., naphthylmethyl or 2-(benzothiazol-2'-yl)ethyl; a saturated or unsaturated cycloalkyl group, e.g., cyclohexyl or cyclohexenyl; a N, O, or S heteroatom containing group, e.g, 4-hydroxybutyl, methoxyethyl, ethoxyethyl or polyalkyleneoxyalkyl; or an aryl group, any of which may be fused to Y such that the emitting fragment contains a lactone ring, or an enzymatically cleavable group containing a bond cleavable by an enzyme to yield an electron-rich moiety bonded to the dioxetane ring; preferably, X is a methoxy group.
The symbol Y represents a light-emitting fluorophore-forming group, part of a luminescent substance capable of absorbing energy upon decomposition of the 1,2-dioxetane to form an excited state from which it emits optically detectable energy to return to its ground state. Preferred are phenyl, biphenyl, 9,10-dihydrophenanthryl, naphthyl, anthryl, pyridyl, quinolinyl, isoquinolinyl, phenanthryl, pyrenyl, coumarinyl, carbostyryl, acridinyl, dibenzosuberyl, phthalyl, or derivatives thereof.
The symbol Z represents hydrogen (in which case the dioxetane can be thermally cleaved by a rupture of the oxygen-oxygen bond), a chemically cleavable group such as a hydroxyl group, an alkanoyloxy or aroyloxy ester group, a silyloxy group, or an enzyme-cleavable group containing a bond cleavable by an enzyme to yield an electron-rich moiety bonded to the dioxetane ring, e.g., a bond which, when cleaved, yields a Y-appended oxygen anion, a sulfur anion, an amino or substituted amino group, or a nitrogen anion, and particularly an amido anion such as a sulfonamido anion.
One or more of the groups represented by the symbols T, R.sup.3 and Z can also include a substituent which enhances the water solubility of the 1,2-dioxetane, such as a carboxy or carboxy-containing group, e.g., a carboxymethoxy group, a sulfonic acid group, e.g., an aryl sulfonic acid group, or carboxylic acid or sulfonate salts, or a quaternary amino salt group, e.g., trimethylammonium chloride, with any appropriate counterion.
Enzymatically cleavable 1,2-dioxetanes can be cleaved using an enzyme such as an alkaline phosphatase that will cleave a bond in, for example, a Z substituent such as a phosphate monoester group, to produce a Y oxyanion of lower oxidation potential that will, in turn, destabilize the dioxetane and cleave its ring oxygen-oxygen bond. Alternatively, catalytic antibodies may be used to cleave the Z substituent. Destabilization can also be accomplished by using an enzyme such as an oxido-reductase enzyme that will cleave the oxygen-oxygen bond directly.
Z in formula I above can also be an enzyme-cleavable alkanoyloxy group, e.g., an acetate ester group, an oxacarboxylate group, or an oxaalkoxycarbonyl group, a 1-phospho-2,3-diacylglyceride group, a 1-thio-D-glucoside group, an adenosine triphosphate analog group, adenosine diphosphate analog group, adenosine monophosphate analog group, adenosine analog group, .alpha.-D-galactoside group, .beta.-D-galactoside group, .alpha.-D-glucoside group, .beta.-D-glucoside group, .alpha.-D-mannoside group, .beta.-D-mannoside group, .beta.-D-fructofuranoside group, .beta.-D-glucosiduronate group, an amide group, a p-toluene sulfonyl-L-arginine ester group, or a p-toluene sulfonyl-L-arginine amide group.
The new synthetic method for producing 1,2-dioxetanes disclosed and claimed in our copending application Ser. No. 402,847 can be illustrated in part by the following reaction sequence leading to the preparation of 1,2-dioxetanes having both an alkoxy (or aryloxy) and an aryl substituent at the 4-position, the latter (illustrated here as an aryl Y substituent) itself being substituted by one or more X.sup.1 groups, these X.sup.1 substituents being ortho, meta, or para to each other. Groups R.sup.2 and X.sup.1 need not be static during the reaction sequence, but may be interconverted under conditions which are compatible with structural considerations at each stage. ##STR3##
In these formulas: T is as described above for Formula I. Any Q can be, independently, a halogen, e.g., chlorine or bromine, or OR.sup.1 ; R.sup.1 can be, independently, a trialkylsilyl group, or an alkyl group, the alkyl group in either case having from 1 to about 12 carbon atoms, and preferably methyl, ethyl, propyl, or butyl; R.sup.2 can be a hydroxyl group, an (ER.sup.4) group, i.e., an ether (OR.sup.4) or a thioether (SR.sup.4) group wherein R.sup.4 is a substituted or unsubstituted alkenyl, alkyl or aralkyl group having up to 20 carbon atoms such as methyl, allyl, benzyl, or o-nitrobenzyl. R.sup.2 can also be an acyloxy group such as acetoxy, pivaloyloxy, or mesitoyloxy, a halogen atom, e.g., chlorine or bromine, a nitro group, an amino group, a mono or di(lower)alkylamino group or its acid salt wherein each lower alkyl substituent contains up to 7 carbon atoms, such as methyl, ethyl, or butyl, where any or all of these lower alkyl groups may be bonded to Y to generate one or more fused rings, or a NHSO.sub.2 R.sup.5 group wherein R.sup.5 is methyl, tolyl, or trifluoromethyl. R.sup.2 can also be a substituted aryl, heteroaryl, or 62 -styrenyl group containing up to 20 carbon atoms such as a 4-methoxyphenyl or 6-methoxybenzthiazol-2-yl group.
R.sup.3 can be a substituted or unsubstituted alkyl, aralkyl, or heteroaralkyl group having up to 20 carbon atoms such as methyl, trifluoroethyl, or benzyl, an aryl or heteroaryl group having up to 14 carbon atoms which may be further substituted, e.g., a 4-chlorophenyl group, a (lower)alkyl-OSiX.sub.3 group in which the lower alkyl group contains up to 6 carbon atoms, such as ethyl, propyl, or hexyl, and any X is independently methyl, phenyl, or t-butyl, an alkoxy(lower)alkyl group such as ethoxyethyl or ethoxypropyl, a hydroxy(lower)alkyl group having up to 6 carbon atoms such as hydroxyethyl, hydroxybutyl or hydroxyhexyl, or an amino(lower)alkyl or mono or di(lower)alkylaminoalkyl group where each lower alkyl group contains up to 7 carbon atoms, such as methyl, ethyl, or benzyl.
X.sup.1 can be hydrogen or a substituted or unsubstituted aryl, aralkyl, heteroaryl, or heteroaralkyl group having up to 20 carbon atoms such as a 4,5-diphenyloxazol-2-yl, benzoxazol-2-yl or 3,6-dimethoxy-9-hydroxyxanthen-9-yl group, an allyl group, a hydroxy(lower)alkyl group having up to 6 carbon atoms such as hydroxymethyl, hydroxyethyl or hydroxypropyl, a (lower)alkyl-OSiX.sub.3 group wherein the alkyl and X radicals are as defined above, an ether (OR.sup.4) or a thioether (SR.sup.4) wherein R.sup.4 is as defined above, an SO.sub.2 R.sup.6 group wherein R.sup.6 is methyl phenyl or NHC.sub.6 H.sub.5, a substituted or unsubstituted alkyl group containing up to 7 carbon atoms such as methyl, trifluoromethyl or t-butyl, a nitro group, a cyano group, an aldehydic function or its oxime or dimethylhydrazone, an alkyl halide group having up to 6 carbon atoms whose halo substituent is preferably chlorine or bromine, a halogen atom, a hydroxyl group, a carboxyl group or a salt, ester or hydrazide derivative thereof, a tri-substituted silicon-based group such as a trimethylsilyl group, or a phosphoryloxy (phosphate monoester) group.
Step 1 of the foregoing reaction sequence involves the formation of a tertiary phosphorous acid alkyl ester from a phosphorous trihalide, e.g., phosphorous trichloride or dialkylchlorophosphite, and an alcohol, e.g., a short chain alkyl alcohol, preferably one having up to 7 carbon atoms such as methanol, ethanol or butanol, in the presence of a base such as triethylamine. An alkali metal alcoholate or trialkylsilanolate can also be used in a direct reaction with the chlorophosphite.
Step 2 involves reacting an aryl aldehyde or heteroarylaldehyde with an alcohol, R.sup.3 OH, to give the corresponding aryl aldehyde acetal. The aryl aldehyde can be a benzaldehyde, a naphthaldehyde, an anthraldehyde and the like. The R.sup.2 substituent on the aryl aldehyde, preferably positioned meta to the point of attachment of the aldehydic group in the benzaldehydes illustrated above, can be an oxygen-linked functional group, e.g., an ester group such as pivaloyloxy, acetoxy and the like, an ether group such as methoxy, benzyloxy, and the like, a nitro group, a halogen atom, or hydrogen (see Tables 2-6 in the above-mentioned copending application). Functional group X.sup.1 in the aryl aldehyde may be located ortho, meta or para to the point of attachment of the aldehydic group to the aryl ring, and can be a lower alkoxy group such as methoxy, ethoxy or the like, hydrogen, or an alkyl group (see Table 2 in the above-mentioned copending application). In the alcohol reactant R.sup.3 OH, R.sup.3 can be, for example, a lower alkyl group such as methyl, ethyl and the like, a lower aralkyl group, a lower alkoxy alkyl group, a substituted amino alkyl group, or a substituted siloxy alkyl group (see Tables 2-6 in the above-mentioned copending application). Diols such as ethylene glycol or propylene glycol, e.g., HO--(CH.sub.2).sub.n --OH, produce cyclic acetals which are within the scope of this invention. The acetalization reaction between the aryl aldehyde and the alcohol or diol is carried out in conventional fashion, preferably in the presence of a catalyst, e.g., a Lewis acid, such as hydrochloric acid, p-toluenesulfonic acid or its polyvinylpyridine salt, or Amberlyst XN1010 resin, accompanied by removal of water. Water can be removed using, e.g., trialkylorthoformate, 2,2-dialkoxypropane, anhydrous copper sulfate, or a molecular sieve, or by azeotropic distillation in, for example, a Dean-Stark apparatus. In cases in which acetalization may proceed with poor conversion or yield, it is possible to use the Noyori reaction wherein any of the aforementioned alcohols (R.sup.3 OH) or diols are reacted with the aldehyde as their mono or bis trialkylsilyl ethers in the presence of trimethylsilyl triflate as catalyst in a chlorinated hydrocarbon solvent.
Step 3 involves reacting the tertiary phosphorous acid alkyl ester (trialkylphosphite) produced in Step 1 with the aryl aldehyde dialkyl or cyclic acetal produced in Step 2, preferably in the presence of at least one equivalent of a Lewis acid catalyst such as BF.sub.3 etherate or the like, to give the corresponding phosphonate, essentially according to Burkhouse, et al., Synthesis, 330 (1984). Aryl aldehyde dialkyl acetals react with between 1 and 1.5 equivalents of a trialkylphosphite in the presence of a Lewis acid in an organic solvent such as methylene chloride, under an inert atmosphere, e.g., argon gas, at temperatures below 0.degree. C., to produce in almost quantitative yields the corresponding 1-alkoxy-1-arylmethane phosphonate esters. The phosphonates are generally oils that can be used directly or purified by chromatography on silica gel or by distillation in Vacuo. .sup.1 HNMR spectra will exhibit a doublet near 4.7 ppm (J=15.5 Hz) due to the benzylic proton, split by the adjacent phosphorous anion; occasionally, two doublets of unequal intensity will be observed.
In step 4, the phosphonate-stabilized carbanion is used to synthesize olefins by the Horner-Emmons reaction. Specifically, in Step 4.1 a phosphonate-stabilized carbanion is produced from a dialkyl 1-alkoxy-1-arylmethane phosphonate in the presence of a base such as sodium hydride, a sodium amide, a lithium dialkyl amide such as lithium diisopropylamide (LDA), a metal alkoxide, or, preferably, n-butyllithium, in a suitable solvent, preferably in the presence of a slight excess of base, e.g., about 1.05 equivalents for each ionizable group present. Suitable solvents for the reaction can have an appreciable range of polarities, and include, for example, aliphatic hydrocarbons such as hexanes, aromatic hydrocarbons such as benzene, toluene and xylene, ethers such as tetrahydrofuran (THF) or glymes, alkanols such as ethanol and propanol, dimethylformamide (DMF), dimethylacetamide, and dimethylsulfoxide, and the like, or mixtures of these solvents. As lithiophosphonates are insoluble in diethylether, but soluble in ethers such as THF, reactions using LDA or n-butyllithium are preferably run in dry THF/hexane mixtures. It is also preferred to carry out the reaction in an inert atmosphere, e.g., under argon gas. At temperatures below 0.degree. C., the reaction of n-butyllithium with phosphonates proceeds rapidly, as indicated by the instantaneous formation of a dark yellow to burgundy colored solution, depending upon the particular phosphonate used and its concentration.
In Step 4.2, the phosphonate-stabilized carbanion is reacted, preferably in molar excess, with a carbonyl compound T=O. When T=O is a substituted or unsubstituted adamantanone, e.g., adamantanone itself, the reaction begins immediately upon addition of the ketone, preferably from about 0.8 to about 0.95 equivalents of the ketone, to the stabilized carbanion, and goes to completion under reflux conditions in from about 2 to about 24 hours. Optimization of the T=O equivalency in each case allows complete conversion of this expensive component.
In Step 5 the enol ether is oxidized. Oxidation is preferably accomplished photochemically by treating the enol ether with singlet oxygen (.sup.1 O.sub.2) to add oxygen across the double bond and create the 1,2-dioxetane ring. Photochemical oxidation is preferably carried out in a halogenated solvent such as methylene chloride or the like. .sup.1 O.sub.2 can be generated using a photosensitizer, such as polymer bound Rose Bengal (Hydron Labs, New Brunswick, N.J.) and methylene blue or 5, 10, 15, 20-tetraphenyl-21H,23H-porphine (TPP). Chemical methods of dioxetane formation, using triethylsilylhydrotrioxide, phosphite ozonides, or triarylamine radical, radical cation mediated one-electron oxidation in the presence of .sup.3 O.sub.2, can also be utilized.
When the oxygen-linked functional group R.sup.2 on the aryl ring of the enol ether is an alkoxy group or pivaloyloxy group, it can be converted to an enzyme-cleavable group such as a phosphate group, an acetoxy group, an O-hexopyranoside group, or the like, by carrying out the following additional steps, involving the enol ether produced in Step 4 of the foregoing reaction sequence, prior to carrying out the oxidation reaction of Step 5, as shown below: ##STR4##
Step 6a involves phenolic ether or thioether cleavage of the R.sup.7 substituent (wherein R.sup.7 is preferably lower alkyl, e.g., methyl, lower alkenyl, e.g., allyl, or aralkyl, e.g., benzyl), preferably with sodium thioethoxide, in an aprotic solvent such as DMF, NMP, or the like, at temperatures from about 120.degree. C. to about 150.degree. C. Cleavage can also be accomplished with soft nucleophiles such as lithium iodide in refluxing pyridine, sodium cyanide in refluxing DMSO, or sodium monosulfide in refluxing N-methyl-2-pyrrolidone. When R.sup.7 is pivaloyl, ester cleavage can be accomplished with NaOMe, KOH or K.sub.2 CO.sub.3 in an alcoholic solvent such as MeOH at temperatures from about 25.degree. C. to reflux (Step 6b).
Acylation of the phenolic hydroxyl group in the thus-obtained hydroxy compound is carried out in Step 7 by adding a small equivalent excess of an acid halide or anhydride, acetic anhydride, or oxalyl chloride with Lewis base, e.g., triethylamine, in an aprotic solvent.
The substituent Q on the cyclic phosphorohalidate used in Step 8 is an electronegative leaving group such as a halogen. The monovalent cation M.sup.+ of the cyanide used in Step 9 can be a metallic or alkali metal cation such as Na.sup.+ or K.sup.+, or a quaternary ammonium cation. The cation B.sup.+ of the ammonium base of Step 10 is an ammonium cation; however, NaOMe can also be used as the base. T, R.sup.3 and X.sup.1 are as defined above.
Steps 8, 9 and 10 can be performed separately or in a one-pot or two-pot operation. A cyclic phosphorohalidate, e.g., cyclic phosphorochloridate, is preferred for use in Step 8 not only because of its monofunctionality, chemoselectivity and enol ether-compatible deprotection mode of action, but also because it is 10.sup.6 times more reactive than the corresponding acyclic compounds. In a 3-step, 2-pot operation, the phenolic hydroxyl group in the free hydroxyl product produced in Step 6 is reacted with 2-halo-2-oxo-1,3,2-dioxaphospholane to yield the cyclic phosphate triester (Step 8). This triester is subjected to ring opening with MCN (e.g., NaCN) to yield the corresponding 2-cyanoethyl diester (Step 9). A base, e.g., ammonium hydroxide or sodium methoxide, then provokes a facile .beta.-elimination reaction, yielding a filterable disodium sodium ammonium salt (Step 10). In benzene, THF, diethylether or DMF, phosphate triester formation induced by a Lewis base (e.g., a tertiary amine such as triethylamine) or with a preformed alkali metal salt or the phenolic enolether can be effected with phosphorohalidates over a temperature range of about -30.degree. to about 60.degree. C. Subsequently, if a pure monosodium cyanoethylphosphate ester is desired, the ring cleavage with alkalicyanide (MCN) in DMF or DMSO can be carried out in a narrow temperature range of between about 15.degree. and about 30.degree. C. However, in a one-pot or in situ mode this is not as important, and the temperature range widens to about 60.degree. C. on the high end.
Aryl phosphate disalts can also be made from the aryl alcohol enol ether product of Step 6 using an activated phosphate diester of the general formula: ##STR5## wherein Q is as described above, and R.sup.8 and R.sup.9 are each independently --CN, --NO.sub.2, arylsulfonyl, alkylsulfonyl or trimethylsilyl. Alternatively, the phosphate triester may contain two trimethyl silyl groups, linked to the phosphorous, as shown in the formula above. This reaction can be carried out in the presence of a Lewis base in an aprotic solvent, and yields an aryl phosphate triester. The triester can then be hydrolyzed with a base, M.sup.+ OH.sup.-, M.sup.+- OCH.sub.3 or M.sup.+ F.sup.-, wherein the cation M.sup..sup.+ is an alkali metal, NR.sup.10.sub.4.sup.+ wherein R.sup.10 is hydrogen or a C.sub.1 -C.sub.7 alkyl, aralkyl, aryl or heterocyclic group, to give the corresponding arylphosphate monoester disalt via .beta.-elimination or simple hydrolysis. Dioxetane formation by reacting singlet oxygen (.sup.1 O.sub.2) with these enol ether phosphate triesters, followed by similar base-induced deprotection to the dioxetane phosphate monester, may also be carried out.
An alkoxy group on the aryl ring of the enol ether can be converted to a D-sugar molecule linked to the ring via an enzyme cleavable glycosidic linkage by reacting the phenolic precursor in an aprotic organic solvent under an inert atmosphere with a base, such as NaH, and then with a tetra-O-acetyl-phexopyranosyl halide to produce the aryl-O-hexopyranoside tetraacetate (Step 11). The protective acetyl groups can then be hydrolyzed off using a base such as NaOCH.sub.3, K.sub.2 CO.sub.3, or NH.sub.3 gas, in an alcohol such as methanol, first at 0.degree. C. and then at 25.degree. c for 1 to 10 hours (Step 12), leaving a hexosidase-cleavable D-hexopyranosidyl moiety on the aryl ring.
When the enol ether aryl phosphates are oxidized to bis-quaternary ammonium or corresponding 1,2-dioxetanes (Step 5 above), ion exchange to a bis-quaternary ammonium or monopyridinium salt allows the facile photooxygenation of 0.06M chloroform solutions in the presence of, preferably, methylene blue or TPP, at cold temperatures, e.g., about 5.degree. C. Slower reaction rates and increased photolytic damage to the product may occur with the use of solid phase sensitizers such as polymer-bound Rose Bengal (Sensitox I) or methylene blue on silica gel.
Aryl monoaldehydes or heteroaryl monoaldehydes other than those coming within the formula: ##STR6## wherein X.sup.1 and R.sub.2 are as defined above, can also be used as starting materials in carrying out the above-described reaction sequence. Included among such aryl monoaldehydes are the polycyclic aryl monoaldehydes having the formula: ##STR7## wherein R.sup.2 is as defined above and is preferably positioned so that the total number of ring carbon atoms separating the ring carbon atom to which it is attached and the ring carbon atom to which the aldehyde group is attached, including the ring carbon atoms at the points of attachment, is an odd whole number, preferably 5 or greater; see Edwards, et al., U.S. patent application Ser. No. 213,672.
Fused heterocyclic acetals or hemiacetals can also be used as starting materials in carrying out the above-described reaction sequence. Included among such fused heterocyclic acetals are those having the formulas: ##STR8## and the like, wherein R.sup.2 is as described above, and W can be OR.sup.3, wherein R.sup.3 is described above, or OH, and n is an integer greater than zero.
Purification of the thus-obtained water-soluble dioxetanes is best achieved at alkaline pH values, e.g., about 7.5 to about 9.0, using reverse phase HPLC with an acetonitrile-water gradient, followed by lyophilization of the product; see Edwards et al., U.S. patent application Ser. No. 244,006.
Typical enzymatically-cleavable water-soluble chemiluminescent 1,2-dioxetanes for use in bioassays which can be prepared by the new synthetic method described in the above-mentioned copending application are the 3-(2'-spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy)phenyl-1,2-dioxetane salts represented by the formula: ##STR9## wherein M.sup.+ represents a cation such as an alkali metal, e.g. sodium or potassium, or a C.sub.1 -C.sub.18 alkyl, aralkyl or aromatic quaternary ammonium cation, N(R.sup.10).sub.4.sup.+, in which each R.sup.10 can be alkyl, e.g., methyl or ethyl, aralkyl, e.g., benzyl, or form part of a heterocyclic ring system, e.g., N-methylpyridinium, a fluorescent onium cation, and particularly the disodium salt. A more systematic name for the latter is 3-(4-methoxyspiro[1,2-dioxetane-3,2'-tricyclo[3.3.1.1.sup.3,7 ]decan]4-yl)phenylphosphate disodium salt.